The proposed research will be a systematic investigation of the molecular mechanism of E. coli DNA-dependent RNA polymerase. A large part of the work will employ bacteriophage lambda DNA as the template for in vitro RNA transcription. Following basic work, which will include binding studies and chain initiation and elongation kinetics, more detailed questions will be asked about the RNA polymerase-promoter interaction. The above studies will answer many questions about the mechanism of RNA polymerase. In addition, we will have established a good system for investigating the effects of the many biological factors which are known to stimulate or inhibit RNA transcription. This work will have direct application to the regulation of in vivo RNA synthesis. We will also apply transient state kinetic methods to this problem using lambda DNA and several synthetic, repeating deoxypolynucleotides as templates. This work will employ the chemical-quench-flow and fluorescence stopped-flow techniques. These experiments will aim to elucidate the detailed mechanism by which RNA is polymerized as accurately as it is. Furthermore, we want to explain the translation of RNA polymerase on a DNA template in biochemical terms. The ultimate objective will be to elucidate those aspects of RNA transcription and its control which are common to both procaryotic and eucaryotic organisms. The proposed work will develop techniques and increase our understanding of RNA synthesis so that we will be better able to study the more complex process of RNA transcription in higher organisms.